2.1 tRNA Production
Maturation and maintenance of tRNA within eucaryal cells requires several processing events including 5′ and 3′ end-trimming, modification of specific bases and in some cases, intron removal. The enzymes for these various steps in processing have been characterized in the yeast, archaeal, mammalian and bacterial systems (Deutscher, M. P. tRNA Processing Nucleases, in tRNA:Structure, Biosynthesis and Function, D. Soll and U. RjaBhandary (eds.), American Society for Microbiology, Washington D.C., (1995), pp. 51-65). 5′ end trimming requires the activity of Rnase P and 3′ end trimming requires the function of various endo- and exo-nucleases. Modification occurs through interaction of tRNA with various modification enzymes. Most tRNAs contain a number of global as well as, species-specific modifications (Bjork, G. Biosynthesis and Function of Modified Nucleosides, in tRNA: Structure, Biosynthesis and Function, D. Soll and U. RajBhandary (eds.), American Society for Microbiology, Washington D.C., (1995), pp. 165-205). In archaea and eucarya, several isoaccepting groups of tRNA contain intervening sequences ranging in size from 14-105 nucleotides (Trotta, C. R. and Abelson, J. N. tRNA Splicing: An RNA World Add-On or an Ancient Reaction? In RNA World II, Tom Cech, Ray Gesteland and John Atkins (eds.), Cold Spring Harbor Laboratory Press (1999) and Abelson et al., 1998, Journal of Biological Chemistry 273:12685-12688). Removal of the intron requires the activity of 3 enzymes. In the first step, the tRNA is recognized and cleaved at the 5′ and 3′ junction by the tRNA splicing endonuclease. The archaeal and eucaryal tRNA endonuclease are evolutionary conserved enzymes and contain a similar active site to achieve cleavage at the 5′ and 3′ splice sites. However, they have diverged to recognize the tRNA substrate in a different manner. The archaeal enzyme recognizes a conserved intronic structure known as the bulge-helix-bulge. This structure is comprised of two 3-nucleotide bulges separated by a 4-nucleotide helix. Cleavage occurs within each bulge to release the intron. The eucaryal endonuclease recognizes the tRNA substrate in a mature domain dependent fashion, measuring a set distance from the mature domain to the 5′ and 3′ splice sites (Reyes et al., 1988, Cell 55:719-730). It has recently been demonstrated, however, that the eucaryal enzyme requires a bulge at each splice site and that the enzyme has actually retained the ability to recognize tRNA by an intron-dependent recognition mechanism identical to that of the archaeal endonuclease (Fruscoloni et al., 2001, EMBO Rep 2:217-221). Once cleaved, the tRNA half molecules are ligated by the action of a unique tRNA splicing ligase (Trotta, C. R. and Abelson, J. N. tRNA Splicing: An RNA World Add-On or an Ancient Reaction? In RNA World II, Tom Cech, Ray Gesteland and John Atkins (eds.), Cold Spring Harbor Laboratory Press (1999) and Abelson et al., 1998, Journal of Biological Chemistry 273:12685-12688). In yeast, the product of ligation is a tRNA with a phosphate at the splice junction. Removal of the phosphate is carried out by a tRNA 2′-phosphotransferase to yield a mature tRNA product (Trotta, C. R. and Abelson, J. N. tRNA Splicing: An RNA World Add-On or an Ancient Reaction? In RNA World II, Tom Cech, Ray Gesteland and John Atkins (eds.), Cold Spring Harbor Laboratory Press (1999) and Abelson et al., 1998, Journal of Biological Chemistry 273:12685-12688).
tRNA is an important component in the translational machinery and is quite stable compared to various other protein-based components (elongation factors, amino-acyl synthetases, etc.). tRNA molecules have very long half-lives. Furthermore, like rRNA and ribosomes, tRNA is present in excess within the cytoplasm of actively growing cells (Ikemura, T. and Okeki, H., 1983, Cold Spring Harbor Symp. Quant. Biol. 47:1087-1097). Thus, specific targeting of tRNA molecules allows a selective inhibition of uncontrolled cell proliferation and not cell growth.
2.2 Pre-mRNA Cleavage
Several processing steps are required before eukaryotic mRNA precursors (pre-mRNAs) are exported to the cytoplasm. Pre-mRNA processing includes capping of the 5′ end, splicing, and the generation of a new 3′ end by endonucleolytic cleavage and polyadenylation. Transcription, capping, splicing and 3′ end processing of pre-mRNAs are coupled processes in vivo (reviewed in Barabino and Kelly, 1999, Cell, 99, 9-11; Minvielle-Sebastia and Keller, 1999, Curr. Opin. Cell Biol., 11, 352-357; Zhoa et al., 1999, Microbiol. Mol. Biol. Rev., 63, 405-445; Hirose and Manley, 2000, Genes Dev., 14, 1415-1429; and Proudfoot, 2000, Trends Biochem. Sci., 25, 290-293).
The 3′ end of the pre-mRNAs are generated in a two-step reaction. The pre-mRNA is first cleaved endonucleolytically and the upstream cleavage fragment is subsequently polyadenylated and the downstream cleavage product is subsequently degraded. Six trans-acting factors are required for the in vitro reconstitution of mammalian 3′ end processing, namely CPSF, CstF, CF Im, CFIIm, PAP, PABP2 (reviewed in Wahle and Ruegsegger, 1999, FEMS Micro Rev., 23, 277-295; and Zhoa et al., 1999, Micoboil. Mol. Biol. Rev., 63, 405-445). Cleavage and polyadenylation specificity factor (CPSF) and cleave stimulation factor (CstF) recognize the hexanucleotide AAUAAA upstream and a G/U-rich sequence element downstream of the cleavage site, respectively. In addition, the cleavage complex contains cleavage factors Im (CF Im) and IIm (CF IIm) and poly(A) polymerase (PAP). After the first step, CstF, CF Im and CF IIm are released together with the downstream cleavage fragment. CPSF remains bound to the upstream cleavage product and tethers PAP to the RNA. PAP is the enzyme responsible for the addition of the poly(A) tail in a processing reaction that also requires both CPSF and poly(A)-binding protein II (PABP2).
2.3 Cancer and Neoplastic Disease
Cancer is the second leading cause of death in the United States. The American Cancer Society estimated that in 2001, there would be 1.3 million new cases of cancer and that cancer will cause 550,000 deaths. Overall rates have declined by 1% per year during the 1990s. There are 9 million Americans alive who have ever had cancer. NIH estimates the direct medical costs of cancer as $60 billion.
Currently, cancer therapy involves surgery, chemotherapy and/or radiation treatment to eradicate neoplastic cells in a patient (see, for example, Stockdale, 1998, “Principles of Cancer Patient Management”, in Scientific American: Medicine, vol. 3, Rubenstein and Federman, eds., Chapter 12, Section IV). All of these approaches pose significant drawbacks for the patient. Surgery, for example, can be contraindicated due to the health of the patient or can be unacceptable to the patient. Additionally, surgery might not completely remove the neoplastic tissue. Radiation therapy is effective only when the irradiated neoplastic tissue exhibits a higher sensitivity to radiation than normal tissue, and radiation therapy can also often elicit serious side effects. (Id.) With respect to chemotherapy, there are a variety of chemotherapeutic agents available for treatment of neoplastic disease. However, despite the availability of a variety of chemotherapeutic agents, traditional chemotherapy has many drawbacks (see, for example, Stockdale, 1998, “Principles Of Cancer Patient Management” in Scientific American Medicine, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10). Almost all chemotherapeutic agents are toxic, and chemotherapy can cause significant, and often dangerous, side effects, including severe nausea, bone marrow depression, immunosuppression, etc. Additionally, many tumor cells are resistant or develop resistance to chemotherapeutic agents through multi-drug resistance.
Therefore, there is a significant need in the art for novel compounds and compositions, and methods that are useful for treating cancer or neoplastic disease with reduced or without the aforementioned side effects. Further, there is a need for cancer treatments that provide cancer-cell-specific therapies with increased specificity and decreased toxicity.
Citation of any reference herein is not to be construed as an admission of its availability as prior art.